The development of the vertebrate brain requires complex genetic networks to coordinate myriad processes across time and space, which include cell proliferation, differentiation and migration, along with axon guidance, synapse formation and elimination. Orchestration of these processes results in the assembly of microcircuitry and distributed systems that constitute a functional nervous system. Disruptions in neural development can have a dramatic impact, resulting in complex brain disorders, such as autism spectrum disorders, schizophrenia, mental retardation and epilepsy. Increasingly, genetic data implicate genes involved in axon guidance and synaptogenesis in these complex brain disorders. In addition, evidence suggests a common etiology for these disorders, as mutations associated with one disorder are also associated with increased incidence of others. Among the molecules that have been implicated in these complex brain disorders are the protocadherins. In particular, mutations in pcdh19 result in a female-limited form of infant-onset epilepsy. Despite the clear role for these molecules in neural development and the etiology of neurodevelopmental dysfunction, relatively little is known about their function in vivo. Here, we propose to generate lines of zebrafish in which pcdh7a, pcdh9, pcdh17 and pcdh19 have been inactivated using zinc finger nucleases (ZFNs). In addition, we propose to generate BAC transgenic lines expressing GFP under the control of the pcdh7a, pcdh9, pcdh17 and pcdh19 regulatory elements. To study the effects of the protocadherin mutations on neural development, we will cross the GFP lines into the mutant backgrounds and use in vivo 2-photon microscopy to determine the impact of the mutations on nervous system development. This work will significantly advance our understanding of protocadherin function, providing a foundation for more mechanistic studies and facilitating a better understanding of how disruption of protocadherins can lead to complex brain disorders. PUBLIC HEALTH RELEVANCE: This project is relevant to human health, as mutations in pcdh19 cause a female-limited form of epilepsy and are associated with an increased incidence of other brain disorders. In addition, pcdh7, pcdh9 and pcdh17 have been implicated in autism or schizophrenia.